Project 3 Abstract The development of an immune response to factor VIII (FVIII) in ~30% of persons with severe hemophilia A (HA) represents the most important complication of treating this condition. The formation of anti-FVIII antibodies (inhibitors) significantly compromises the clinical care of these patients, and their subsequent management is less predictable, practically challenging and often extraordinarily costly. The pathogenesis of FVIII inhibitors is recognized to be complex and multifactorial. Most patients that develop this complication do so during the first 25 exposures to FVIII replacement therapy, within the first 2-3 years of life. While there are clear genetic predisposing factors for inhibitor development, such as the mutant F8 genotype, a range of acquired influences also contribute to inhibitor risk. In this project, we will investigate the role of an environmental factor that, to date, has not been studied in this context ? the gut microbiome. There is substantive evidence that the gut microbiome plays an important role in the development and regulation of the systemic immune system, and that there are clear associations between the gut microbiota and certain forms of immunopathology. In addition, the microbiome undergoes significant changes during the first two years of life, the time when initial FVIII treatment occurs in boys with severe HA, and the period of maximum risk for FVIII inhibitor generation. In Project 3 of this research program we will address three objectives related to microbiome influences on FVIII inhibitor formation. We will first evaluate the influence of changes to the gut microbiome-related metabolite environment on the immunogenicity of FVIII. In particular, we will determine whether the products of tryptophan metabolism and levels of the short chain fatty acids, acetate, butyrate and propionate influence innate and adaptive immune responses to intravenously administered FVIII. In the second series of experiments, we will conduct studies utilizing our recently developed germ-free HA mouse model. In these studies, we will begin with an assessment of the likelihood for FVIII inhibitor development in the absence of gut microbes, and will then progress to studies in which we selectively repopulate the gut microbiota through various strategies including fecal transplantation from HA mice with and without FVIII inhibitors. Lastly, we will conduct studies to evaluate the role of the gut microbiome in regulating the immune response to orally delivered FVIII. These experiments will assess the interaction of FVIII with various antigen presenting cell populations and will determine the cytokine and T cell context of the FVIII immune response in the gut associated lymphoid tissue, liver and spleen. Overall, we propose that this program of research will provide important new knowledge concerning the role of the gut microbiome and FVIII immunogenicity. This new information is very likely to have significant biological and potential translational implications.